Protocols in Current Issue
    Intracellular IRF5 Dimerization Assay
    Authors:  Cherrie D. Sherman and Betsy J. Barnes, date: 05/20/2021, view: 810, Q&A: 0

    The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5

    Imaging Microtubules in vitro at High Resolution while Preserving their Structure
    Authors:  Camille Cuveillier, Yasmina Saoudi, Isabelle Arnal and Christian Delphin, date: 04/05/2021, view: 468, Q&A: 0

    Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection

    Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
    Authors:  Shaista Anwer and Katalin Szaszi, date: 07/20/2020, view: 1372, Q&A: 0
    [Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows ...
    Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures
    Authors:  Johanna M. Schafer and Jennifer A. Pietenpol, date: 07/05/2020, view: 1505, Q&A: 0
    [Abstract] MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system ...
    Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
    Authors:  Terra M. Kuhn, Shawn C. Little and Maya Capelson, date: 07/05/2020, view: 4185, Q&A: 0
    [Abstract] Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 1368, Q&A: 0
    [Abstract] Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and ...
    FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells
    Authors:  Brandon T. Milliken, Robert P. Doyle, George G. Holz and Oleg G. Chepurny, date: 06/05/2020, view: 2461, Q&A: 0
    [Abstract] Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca2+. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors ...
    Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
    Authors:  Balaji T. Moorthy, Akhilesh Kumar, Lauren X. Lotenfoe and Fangliang Zhang, date: 05/20/2020, view: 2145, Q&A: 0
    [Abstract] In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short ...
    Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images
    Authors:  Akira Kitamura and Masakata Kinjo, date: 05/20/2020, view: 1900, Q&A: 0
    [Abstract] Size determination of subcellular structures such as inclusion bodies (IBs) and granules from fluorescent images is important for identification and structural characterization. However, it is often time-consuming just for the comparison of the average size of the structures. Here, we introduce a high-throughput procedure to represent the average ...
    Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
    [Abstract] CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and ...

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